The largest tissue of human body is skeletal muscle, which is responsible for voluntary movements and breathing. Muscular dystrophies are a group of debilitating inherited diseases, characterised by progressive skeletal muscle wasting, followed by accumulation of fat and connective tissue. Current standards of care do not provide effective treatment, and can only delay loss of ambulation, cardiac and respiratory problems. Some muscular dystrophies cause premature death.
Duchenne muscular dystrophy (DMD) is the most common and currently incurable neuromuscular disorder. Numerous animal models have been developed for DMD, including the most commonly used mdx/dmd mice and larger animals, such as dogs and pigs. However, the mdx/dmd mice do not fully recapitulate human pathophysiology. Unfortunately, many drugs that could ameliorate phenotypes in mdx/dmd mice fail to show efficacy in clinical trials. To replace/reduce the use of mouse models in muscular dystrophy research, we propose to develop human-specific, physiology-relevant in vitro models that can be exploited for elucidating disease mechanisms and testing drug candidates for developing novel therapies. However, human primary myoblasts lose myogenicity after extensive expansion in culture and lack isogenic control cells for comparison.
To overcome these challenges, we generated two iPSC lines from two patients with distinct DMD mutations. Using CRISPR-Cas9 genome editing, we precisely corrected the DMD mutations to obtain two CRISPR-corrected iPSC lines as isogenic controls. Following a transgene-free myogenic differentiation protocol, the isogenic pairs of iPSC lines were differentiated to myogenic progenitors, resembling human primary muscle precursor cells. Terminal differentiation of human iPSCs-derived myogenic progenitors formed multinucleated, striated myofibers. Full-length dystrophin expression was completely restored in the CRISPR-corrected muscle cells. As standard 3D culture does not reflect the complexity of highly aligned myofiber architecture in vivo, we propose to employ novel bioengineering technologies to bridge this gap. We will use 3D bio-printing to fabricate human iPSC-derived skeletal muscle constructs, followed by characterisation of the biophysical and biological properties of the 3D bio-printed human skeletal muscle constructs. We will establish a range of disease-relevant functional assays by assessing isogenic pairs of bio-printed human 3D models, such as contractile force generation, response to chemical and electric stimulus, oxidative stress, as well as calcium handling. Finally, as proof of concept, we will test candidate drugs in our physiology-relevant 3D in vitro models to investigating their efficacy in ameliorating pathophysiological phenotypes.
In brief, human iPSC-derived myogenic progenitors in combination with 3D bio-printing technologies can provide human-specific platforms critically needed for elucidating disease mechanisms and facilitating drug discovery. Importantly, our experimental paradigms are broadly applicable to any muscle disease. This multidisciplinary project will have a significant impact on developing novel human pre-clinical models and replacing/reducing the use of mouse models in muscular dystrophy research.